The proposed research in this renewal application seeks to understand what roles CFTR plays in the function of human airway epithelium, and how its malfunction in CF leads to the airway pathology characteristic of this disease. Five interrelated projects will study aspects of CFTR ranging from the molecular to the clinical. Dr. Lingappa (Project 1) will study how CFTR is inserted into cell membranes and is trafficked within the cell. Dr. Verkman (Project 2) will study the function of CFTR in intracellular organelles. In particular, using newly developed techniques, he will test the conclusions of others that CFTR is involved in exocytosis and in regulating the pH of endosomes. Using the patch-clamp technique, Dr. Wine (Project 3) will characterize in detail the anion channel properties of wild-type and mutant CFTR. Recent work from our Center has demonstrated that mediator-induced Cl secretion by cultures of CF airway submucosal gland cells is less than 5% of normal. Therefore, the aim of Project 4 (Widdicombe & Finkbeiner) will be to test the hypothesis that the initial accumulation of airway mucus in CF reflects predominantly dehydration or other alterations in gland mucous secretions. Finally, Dr Szoka (Project 5) will test a non-viral approach towards gene therapy of CF. Attempts will be made to transfect airway epithelium in vivo by both aerosol and intravenous routes. The latter route increases the likelihood of correcting defects in submucosal gland cells. All projects will utilize the highly differentiated primary cultures of surface and gland epithelial cells provided by a cell culture CORE directed by Dr Finkbeiner.